Production of riboflavin by ashbya gossyph



Patented Mar. 10, 1953 PRODUCTION OF RIBOFLAVIN BY ASHBYA GOSSYPII Samuel C. Beesch, Columbus, Ohio, and Melvin C. Firman, Philadelphia County, Pa., assignors to Publicker Industries Inc., Philadelphia, Pa., a corporation of Pennsylvania No Drawing. Application May 25, 1948, Serial No. 29,196

9 Claims.

The present invention relates to a process for the biochemical synthesis of riboflavin. More particularly it is concerned with the production of riboflavin by the action of the fungus known as Ashbya gossypz'z' on a novel type of mash.

The above mentioned organism has previously been employed in the synthesis of riboflavin from liquid media containing animal protein, corn steep liquor, and dextrose. While relatively good yield of riboflavin are obtainable by employing mashes of the aforesaid type it has, in the past, been assumed that a source of animal protein, a relatively expensive ingredient of the mash, was essential for the procurement of satisfactory results. Such animal protein, thought to be essential for successful fermentation, has been supplied in the form of peptone, beef extract, and stick liquor. The utilization of peptones, industrialwise, is costly and in the case of beef extract and stick liquor a preliminary enzymatic hydrolysis with papain is required before such materials can be used, thereby further increasing the operation cost.

It has now been discovered that the animal protein which has heretofore been regarded as an indispensable ingredient of mashes employed in the production of riboflavin by the action of the organism Ashbya gosspyiz' thereon can be replaced entirely by cheap vegetable protein and it is to such vegetable protein containing mashes that the present invention is directed.

In utilizing the novel mashes of the present invention, the fermentation may be effected in accordance with any of several procedures known to the art. A suitable procedure for carrying out the fermentation consists essentially of fermenting with the fungus 'Ashb'ya gossypiz' under pure culture conditions a mash of the type contemplated by the present invention and having a predetermined pH. While fermentation is under way sterile air i introduced in such a manner that the fungus and air are substantially completely dispersed throughout the mash. In order to avoid any possible contamination from the atmosphere during the fermentation a positive operation pressure is preferably maintained over the surface of the mash. Toward the conclusion of the fermentation the pH will ordinarily be found to be between 6.5 and 7.5. After fermentation is complete the mash is filtered by any convenient method and the riboflavin present in the filtrate may be recovered therefrom in accordance with any of several well known methods, for examples, by absorption on a suitable adsorbent material such as fullers earth or adsorbent clay. The resulting 'adsorbate can then be treated in a known manner with a suitable elution solvent, as for example, that described in U. S. Patent No. 2,175,014 to Booher et al., to obtain the riboflavin in a substantially pure state.

The nutrient mashes of the present invention may vary in composition. In addition to the nutritive amount of vegetable protein present there is required from about 60% to about 75% of a metabolizable carbohydrate, based on the total weight of nutrients in the mash, and from about 15% to about 20% of a suitable natural source of amino acids and necessary growth factors such as are found in corn steep liquor. Suitable examples of metabolizable vegetable proteins are soy bean meal, linseed meal, cotton seed meal, citrus pulp, thin grain ethyl stillage (ethyl solubles), i. e., the spent mash from whiskey or yeast ethyl alcohol fermentations, and the like. On the other hand we have found that, at least under the conditions investigated, vegetable proteins such as those obtainable from barley malt, corn and malted corn are unsuitable for riboflavin production utilizing Ashbya gosspyiz' as the fermentative organism. The carbohydrates employed may be any of those previously used in mashes fermented by Ashbya gossypii, such as for example, glucose. r

In general, the total nutrient content ofthe mash may vary from between about 3.5% to about 8%, depending upon the composition of the particular mash utilized. Obviously it is 'desirable to employ mashes having the maximum initial concentration of nutrient substances practicable under the circumstances in order to conserve operating space and equipment. Mashes having a nutrient content appreciably in excess of 8%, however, are generally not suited for satisfactory growth of the organism and, hence from a practicable standpoint, at least, are undesirable. For most purposes it is preferred to employ mashes having an initial nutrient content of about 6% sincemashes of such concentration seem to be favorable both from the standpoint of growth of the organism and relative amounts of riboflavin produced. In regard to the nutrient content of the mash, however, it should be understood that if the amount of nutrient present decreases as the fermentation proceeds, such content may be intermittently increased to the original value by the addition of the required nutrient substances as many times as may be found necessary or desirable under the circumstances.

Aeration of the mash during the fermentation may be accomplished by any desired means, the chief requirements being that the air employed should be sterile and that it be introduced in quantities and in a degree of dispersion sufiicient for'a rapid growth of the fungus. Incertain instancesit inay even be" found desirable to employ both aeration and moderate mechanical agitation in order to facilitate a more rapid and satisfactory growth of the fungus. Frequently, it has been found that such practice results in shorter fermentation periods, and is particularly found to be true when employing relatively small volumes of mash. In the case of larger volumes of mash, however, the organism will generally venient method for aerating comparatively large volumes of such mashes consists of introducing sterileeirfthrough a dispersion unit or 'a 'series of such 'uir'iitsflthereby causing the air to be dividedinto bubbles of small diameter. Although the rate atwliichsuch aeration should occur may "anytime relativelyuvidelimits it has generally been found preferable to introduce it are rate foffi omabout 1.5 to about 2.0 cubic feet per minute- -per square foot "of mash surface. While the atave menuoned inethod'of'aerating the mash has been 'foiindparticularly' effective when em- 9 pl'o'ying" the customary fermentation procedures, it should'be'undrst'ood that the term aeration, as stain the" present description as well as in [the a peared olaiins'jis' not to be restricted to such methods. For'ex'ample, under certain conditions'ample aeration can be effected by stirring, Q spra yifig them'ash inthe presence of' an atmos- -'phere' of sterileall,or bysha'king in an atmosph'ereof sterile'air, the latter method being i1- liistratedin the example which appears below. "The initial pI-I'of the inashes'employedin the pres'ent invention may vary" from between about 'i 5.510 about 715, although for the majority 'of mashes it"ispr'eferable'to utilize'an'initial pH of'fro m about 6.0to"about6.5. On completion for thefrineiitauo 'the' "pn'cut of the mash is generallyiound to be from'ab'out 7.01: v.5. lifgeneral the temperature at 'whi'chfernientation may be effected can varyffrom'about C. Qmacmtevw. However-Jane's beenioune prefe1jable to emp loytempei atures of from about 24 G. to about 29 'C. At tehiperatures below --20 C. the fermentation" 'i'at'e'is reduced to "such ;an extent that onlynegligibleamounts of ribo- -flavin are produced while at temperatures inex- -cess of about-31C. the ability of the organism to; produce riboflavin is greatly impaired.

-The;p'eriod' required to carry fermentation 'to completion is, of course, dependent upon -nu- -merous factors such as temperature, thenutrie'nt content-of the particular mash fermented, the percentage of inoculant'employed, etc. Usually theifermentation will beiifoundtobe complete within-approximately five days after inoculation; however, injany'giveninstance this'time may readily be ascertained by periodic assays of mash s m e e ima ri e e asce The inoculant containing the fungus Ashbya Toma/pantry be preparedin accordanoe'with a number of "dirrerm methods however, we have fouiid'thefollowingprocedure to be quite satisfactory: firstasto'ck'slant of an Ashby a'goss'ypii culftii'it istransfeiired to 'a'sla'nt' composed of autolized of about 1.0%. mashesof the present invention may be satisfactorilyeffected by means of spore inoculation in accordance with the procedure described and -'-claimed in cop'en'ding' application U. S. Serial No.

17,124, filed March 25, 1948, by Samuel C. Beesch and M.'Shon. In accordance with this process large volumes of mash are inoculated with a *sp'ore'inoculumgenerally in a dilution of from about one part of inoculum to 160,000 parts of mash to one part of inoculum to 320,000 parts of mash. This particular procedure is found to be especially advantageous where the organism employed iskn'ownto for'rnmuta'nt strains vvhich producever y {1i ttle riboflavin.

-Whiletheriboflavin present in the liquors resulting i from the fermentation "process 5 of the present" invention-ma be recovered therefrom by means of any ofseveral standard procedures,

same of which have been; previously 'mentioned,

a further advantage of the present invention 'confstitutes; the-fact that dried residues can besecured containing riboflavin in amounts of from between about 800- to 25,000pg.-per gram of dried materiaI-byconcentrating said -liquors in accordance with well established'procedures. However,

""careshould -be e'iercised in "the concentration tress to avoidtemperatures and pH values which would" result in sub'stantial destruction of the "'i'ibcfiavin activity'of the resulting residue. Gen- "'erally, 'the 'residues rich in' riboflavin can be secured by first concentrating the fermented m'a'sh through evaporation, such as for example, "by the use of multiple'eflect evaporators, after which the'resulting' concentrate may be dried to asolid 7 mass by evaporation in pans,- orby'the use of rotar T drum 'driers, 'va'cuum driers, spray driers, -or thelili'e. In "general'it may be'said that any "of the common "methods fori'ecoverin'g solids "afiect the riboflavin present therein, may be'em- "ployed for this 'purpose. "It "will, therefore, "be

evident-that the" abov -mentioned fatnreof the present invention iscapable' of'nuinerous modifications, the only essential-requirements *being that therec'overyst'e'p is carried out in a'n'lann'er not harmful to the'riboflavin cOntainedin'the'resuiting residue.

The present invention may be more specifi'call "illustrated'by the following example:

EXAMPilE In'a"'sries of" experiments, mashesw'eie prepared by' addingthe 5 desired vegetable protein,

corn'steep, and dextrose to distilled-"water i'nithe "coricentrationsindicated below. Control inashes -'were' also prepared in-which peptone; employed in the' standard 'rhashes, was; substituted for the *vegetable protein. The ma shes were'firstads'el'wasfabout 1' men, an -atmosphereofsterile-air being maintaind thereo'ver tat fall times. I The vessel containing the-inoculate dinas'h was-plated on a standard platform shaker and agitated in order to secure abundant aeration and incubated for a period of five days at 27 C. The results appearing below in tabular form show the number of g. of riboflavin per ml. produced from both standard type mashes and mashes in which vegetable protein was substituted for animal protein.

Table Composition of Mash fi gg? 1% corn steep (solids) 1% soy bean meal 2 610 0. 5 7. 2 4% dextrose 1% corn steep (solids) 1% ethyl solubles 3 420 6. 5 7.2 4% dextrose 1% corn steep (solids 1% cottonseed meal l 352 6. 5 7. 5 4% dextrose 1% corn steep (solids) 1% linseed meal 3 310 6. 5 7. 3 4% dextrose 1% corn steep (solids).. 1% orange pulp meal. 1 343 6. 5 7. 2 4% dextrose 1% peptone 1% corn steep (S011 525 6. 5 7. 5 4% dextrose 1 Control mash-average of 4 fermentations. 5 Average of 4 fermentations. 3 Average of 3fermentat1ons.

What we claim is:

1. A process for the production of riboflavin which comprises forming a nutrient mash containing as its principal nutrients a metabolizable carbohydrate, corn steep liquor, and a substantially unhydrolyzed protein selected from the group consisting of ethyl solubles, cottonseed meal, linseed meal, citrus pulp and soy bean meal, to the exclusion of degraded proteinaceous material and of animal protein, and fermenting the mash with the fungus Ashbya gossypii while maintaining the mash in a state of aeration sulficient to disperse the fungus and air throughout the mash.

2. A process according to claim 1 wherein the mash is adjusted to a pH of about 5.5-7.5 prior to the fermentation and wherein the fermentation is carried out at a temperature of about 20-37 C.

3. A process according to claim 1 wherein the unhydrolyzed vegetable protein is soy bean meal.

4. A process according to claim 1 wherein the unhydrolyzed vegetable protein is ethyl solubles.

5. .A process according to claim 1 wherein the unhydrolyzed vegetable protein is cottonseed meal.

6. A process according to claim 1 wherein the unhydrolyzed vegetable protein is linseed meal.

7. A process according to claim 1 wherein the unhydrolyzed vegetable protein is citrus pulp.

8. A process according to claim 1 wherein the mash is adjusted to a pH of about 6-7 prior to fermentation and wherein the fermentation is carried out at a temperature of about 26-30 C. and wherein air is introduced at a rate of about 1.5-2.0 cubic feet per minute per square foot of mash surface.

9. A process for the production of riboflavin which comprises forming a nutrient mash containing as its principal nutrients about -75% of a metabolizable carbohydrate, about 15-20% of corn steep liquor, and about 15-20% of a substantially unhydrolyzed protein selected from the group consisting of ethyl solubles, cottonseed meal, linseed meal, citrus pulp and soy bean meal, to the exclusion of degraded proteinaceous material and of animal protein, said percentages being based on the weight of total nutrients present in the mash, adjusting the pH of the mash to about 5.5-7.5, and fermenting the mash with the fungus Ashbya gossypz'i at a temperature of about 20-37 C. while maintaining the mash in a state of aeration suflicient to disperse the fungus and air throughout the mash.

SAMUEL C. BEESCHV MELVIN C. FIRMAN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,445,128 Tanner July 3, 1948 2,461,922 Rake Feb. 15, 1949 OTHER REFERENCES Tilden, Jour. Bact., 1939, page 629.

Brewer, Jour. Bact., October 1943, pages 395- 396.

McMahon, Jour. Bact., April 1944, pages 400- 401.

Schopfer, Helvetica Chimica, Acta V, XXVII, Aug. 1, 1944, pages 1017-1031.

Wickerham, Arch. Biochem, vol. 9, No. 1, Ja 1946, pages to 98.

Tanner, Jour. Bact., 54 (1947), pages 38-39. 

1. A PROCESS FOR THE PRODUCTION OF RIBOFLAVIN WHICH COMPRISES FORMING A NUTRIENT MASH CONTAINING AS ITS PRINCIPAL NUTRIENTS A METABOLIZABLE CARBOHYDRATE, CORN STEEP LIQUOR, AND A SUBSTANTIALLY UNHYDROLYZED PROTEIN SELECTED FROM THE GROUP CONSISTING OF ETHYL SOLUBLES, COTTONSEED MEAL, LINSEED MEAL, CITRUS PULP AND SOY BEAN MEAL, TO THE EXCLUSION OF DEGRADED PROTEINACEOUS MATERIAL AND A ANIMAL PROTEIN, AND FERMENTING THE MASH WITH THE FUNGUS ASHBYA GOSSYPII WHILE MAINTAINING THE MASH IN A STATE OF AERATION SUFFICIENT TO DISPERSE THE FUNGUS AND AIR THROUGHOUT THE MASH. 